Cholangiocarcinoma is a highly malignant tumor that arises from cholangiocytes of the intra- and extra-hepatic biliary system. Studies from our group and others have found that modulating cell death/apoptosis pathways provide potential novel avenues for cholangiocarcinoma therapy. Down-regulating the death receptor (Fas) and up-regulating Fas Ligand in cholangiocarcinoma cells protect cells from apoptosis and enhance their tumorigenesis in mice. Further, several anti-apoptotic mediators in the Fas signaling pathways, including protein kinase B/AKT and FLICE like Inhibitory Protein (FLIP) are up-regulated in cholangiocarcinoma cells, which render the cancer cells more resistant to apoptosis and divert Fas-induced signaling to survival and proliferative pathways. With human cholangiocarcinoma samples we demonstrated that the expression of Fas is associated with tumor differentiation. We have found that antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells via a mechanism related to the Fas-mediated apoptosis pathways and inhibit tumorigenesis in mice. Mechanistic studies further identified Ca2+dependent direct binding between CaM and Fas, which is regulated upon Fas-activation. In addition, CaM is recruited into the Fas-activated death inducing signaling complex (DISC). A CaM antagonist and a calcium chelator inhibit recruitment of CaM into the Fas-induced DISC and block the recruitment of FADD into the DISC, suggesting that CaM/Fas binding contributes to Fas-activated DISC formation. Recently, we found that CaM binds to FLIP, another DISC protein that is elevated in cholangiocarcinoma cells and activates survival signals upon Fas activation. Therefore, we hypothesize that CaM is a critical regulator of the Fas- death receptor signaling pathway and represents a potential therapeutic target for cholangiocarcinoma. In this application, we will continue pursuing our long-term goal of understanding the Fas death receptor signaling pathways in the pathogenesis of cholangiocarcinoma by focusing on determining the role of CaM in regulating the Fas-activated DISC and the function of CaM/Fas and CaM/FLIP binding in regulating proliferation and apoptosis of cholangiocarcinoma in culture and tumorigenesis in animal models. The Specific Aims are: 1) characterize the function of CaM in regulating Fas signaling pathways in cholangiocarcinoma cells;and 2) characterize the role of the CaM/Fas/FLIP interaction in regulating cholangiocarcinoma tumorigenesis in mice. The present studies will define the fundamental mechanisms by which CaM regulates signaling through the Fas pathway, thus facilitating further studies to translate these findings into strategies and therapies for patient care. Considering that cancer is one of the important health problems in the Veteran's population, our studies will lead to improvement of the health of veterans. PUBLIC HEALTH RELEVANCE: Relevance to Veterans Health: Cancer and specifically hepatobiliary cancer is an important health problem in the Veteran's population. Steps to diagnose and treat patients with this highly fatal disease would be a welcome addition to the arsenal of cancer diagnostic tests and therapeutic agents. Furthermore, the fundamental understanding of the mechanisms responsible for cell death (apoptosis) and their regulation will likely impact treatment of other forms of cancer and other diseases in which altered apoptosis plays an important role such as Alzheimer's disease, Osteoporosis, and Diabetes Mellitus. All of these diseases are very important health problems for the Veteran's population.